Introduction 1 Introduction 1.1 RepA DNA helicase Helicases are ubiquitious motor proteins essential in key biological processes which require single-stranded DNA (ssDNA) such as DNA replication (Fig. 1.1), transcription, repair and recombination (Matson et al., 1990; Lohman et al., 1996). The basic reaction catalyzed by this family of enzymes is the unwinding of the duplex form of DNA or RNA, a process coupled to nucleoside triphosphate (NTP) hydrolysis. The unwinding of double-stranded DNA (dsDNA) by helicases is either in 5´® 3´ or in 3´® 5´ direction. Since the discovery of the first helicase from Escherichia coli (Abdel-Monem et al., 1976), a growing number of helicase proteins which are involved in many aspects of DNA metabolism in bacterial, viral and eukaryotic systems have now been characterized in vitro. In humans, malfunction of certain DNA helicases is associated with several severe diseases including Bloom´s syndrome, Xeroderma Pigmentosum and Werner´s syndrome (Friedberg, 1992; Hanawalt, 1994; Yu et al., 1996), with the development of cancer (Egelman, 1996), and with ageing (Bowles, 1998). Figure 1.1: DNA replication fork. The two ssDNA stands unwound by DNA helicases serve as templates for the DNA polymerase to synthesize new, complementary DNA (leading and lagging strand). - 1 - Introduction The replicative hexameric helicase RepA is encoded by the broad host range plasmid RSF1010, an 8684-base-pair (bp) multicopy plasmid that can replicate in a wide variety of Gram-negative and also Gram-positive actinomyces (Scholz et al., 1989). Three plasmid- encoded proteins, RepA, RepB´, and RepC, exhibit RSF1010-specific helicase, primase and initiator protein activities, repectively, and are essential for the replication of this plasmid. E. coli DNA gyrase, SSB (single strand DNA binding protein) and the production of dnaZ (g subunit of DNA polymerase III holoenzyme) are also required for replication of plasmid RSF1010, while the bacterial RNA polymerase and the DnaA, B, C, G, T proteins are not (Scherzinger et al., 1991). RepA is one of the smallest known homohexameric helicase enzymes with a total molecular mass of 180 kDa (Scherzinger et al., 1997). It unwinds dsDNA in 5’® 3’ polarity and prefers a tailed substrate with an unpaired 3’- tail mimicking a DNA replication fork. The RepA activity is fueled by ATP, dATP, GTP, and dGTP and less efficiently by CTP and dCTP while UTP and dTTP are poor effectors. Optimal unwinding activity was found at a narrow pH range around 5.5 (Scherzinger et al., 1997), similar as observed for yeast Saccharomyces cerevisiae RAD3 helicase (Sung et al., 1988). Below pH 5.6 and at low salts concentration, the RepA hexamers aggregate and form tubular structures (Röleke et al., 1997). 1.2 Structural features of DNA helicase All DNA helicases for which the assembly state of the enzyme has been examined appear to function as oligomers, generally dimers or hexamers, thus providing multiple potential DNA binding sites, which are required for helicase function. The E. coli DnaB protein (San Martin et al., 1995; Yu et al., 1996), the bacteriophage T7 gp4 protein (Egelman et al., 1995), the E.coli branch migration RuvB protein (Stasiak et al., 1994), and the E. coli transcription termination protein Rho (Gogol et al., 1991) assemble as hexamers into a ring shape; by contrast, the E. coli proteins Rep (Wong et al., 1992) and UvrD (Runyon et al., 1993) (Helicase II) have been characterized as dimers. Detailed information is shown in Table1. - 2 - Introduction Table 1.1: Characteristics of DNA and RNA helicases. Protein Family group Molecular Direction of Assembly Minimal weight unwinding state requirement for oligomer formation E.coli bacteriophage DnaB-like 4A 62,655 5' to 3' Hexamer dTTP, dTDP, T7 gp4 4B 55,743 dTMP-PCP, ATP, dATP E. coli bacteriophage DnaB-like 53,601 5' to 3' Hexamer ATP, GTP, T4 gp41 ATPgS,GTPgS RSF1010 RepA DnaB-like 29,909 5' to 3' Hexamer None E. coli DnaB DnaB-like 52,390 5' to 3' Hexamer Mg2+ E. coli RuvB AAA+ family 37,174 5' to 3' Hexamer Mg2+ E. coli rho F1-ATPase 47,004 5' to 3' Hexamer RNA B. subtilis phage DnaB-like 46,746 5' to 3' Hexamer ATP, Mg2+ SPP1 gene 40 Simian virus large SF III 81,907 3' to 5' Hexamer ATP, ADP, T antigen ATPgS Bovine papillomavirus papillomavirus 68,246 3' to 5' Hexamer DNA E1 family Human Bloom's SFII 159,000 3' to 5' Hexamer ATPgS, Mg2+ syndrome helicase Human MCM4 MCM family 96,606 3' to 5' Hexamer Not known E. coli Rep SF I 68,000 3' to 5' Dimer DNA E. coli Helicase II SF I 82,116 3' to 5' Dimer DNA (UvrD) Concerning the primary amino acid sequences, all known helicases have been grouped into families and superfamilies (SF) (Gorbalenya et al., 1993; Ilyina et al., 1992). For example, E. coli Rep, PcrA, and E. coli UvrD belong to superfamily I (SF1); the BLM helicase belongs to the Rec-Q family in SF2; the viral large T antigens belong to the SF3 superfamily. The hexameric helicases do not fall within a single family, but are members of various families. - 3 - Introduction For example, the helicases RepA, DnaB, P1 Ban, SSP1 G40P, T7 gp4, and T4 gp41 belong to the DnaB-like family; and the rho protein belongs to the V- and F1-ATPase family. The smaller family of DnaB-like helicases shows five conserved motifs H1, H1A, H2, H3, H4 (Ilyina et al., 1992). The conserved H1 and H2 motifs contain the Walker A and B sequences (Walker et al., 1982). A highly conserved and essential lysine in H1 and aspartic acid in H2 have been identified from mutagenesis and structural studies. A conserved glutamic acid in H1a has been proposed to play a role in NTP hydrolysis (Sawaya et al.,199; Story et al., 1992; Abrahams et al., 1994). Little is known about the exact role of H3 and H4 may be involved in DNA binding (Washington et al., 1996). Additionally, there are several residues beyond H4 which show some sequence conservation, and they are involved in nucleotide binding and hydrolysis (Patel et al., 2000). Several high-resolution structures have been reported for monomeric/dimeric helicases. A common helicase architecture has been revealed by crystal structures of two DNA helicases, Bacillus stearothermophilus PcrA (Subramanya et al., 1996; Velankar et al., 1999) and E. coli Rep (Korolev et al., 1997), and an RNA helicase, the hepatitis C virus NS3 protein (Yao et al., 1997; Cho et al., 1998; Kim et al., 1997). The recently determined crystal structures of PcrA complexed with a DNA substrate have revealed details of the helicase mechanism (Soultanas et al., 2000). Of the intact, unmodified replicative hexameric helicases, information is only limited to low-resolution electron microscopic studies (Fig. 1.2). Crystal structure analyses of truncated domains of two hexameric helicases, DnaB (Fass et al., 1999) and T7 bacteriophage helicase-primase (Sawaya et al., 1999; Singleton et al., 2000) have been reported. In the structure of hexameric fragment of gene 4 helicase from bacteriophage T7, the deviation from expected six-fold symmetry of the hexamer indicates that the structure represents an intermediate on the catalytic pathway. The structural consequences of the asymmetry suggest a “binding change” mechanism to explain how cooperative binding and hydrolysis of nucleotides are coupled to conformational changes in the ring that most likely accompany duplex unwinding. - 4 - Introduction Figure 1.2: Electron microscopy images of hexameric helicases (Patel et al., 2000). 1.3 Nucleotide binding and NTP hydrolysis of helicases All DNA helicases possess a consensus NTP binding site, as indicated by the presence of the conserved Walker A and B motif (I and II) (Gorbalenya et al., 1993). For helicase-catalyzed unwinding of dsDNA, NTP binding and hydrolysis are essential. The NTPs can function as switches that induce conformational changes necessary to promote DNA binding and release- steps required for translocation of helicase along the DNA double helix (Wong et al., 1992). Until now the molecular mechanism by which NTP binding and hydrolysis are coupled to DNA unwinding is poorly understood. Homo-oligomeric helicases feature at least one potential NTP binding site per subunit and nucleotides appear to bind to the oligomer with negative cooperativity or possibly two classes of “high” and “low” affinity sites (Patel et al., 2000). It is a common feature of the hexameric helicases that the six potential nucleotide binding sites are nonequivalent and can be clearly distinguished as three high affinity and three low affinity binding sites, as found for DnaB helicase (Bujalowski et al., 1993) and for transcription termination protein Rho (Geiselmann et al., 1992). In the hexameric - 5 - Introduction bacteriophage T7 DNA helicase, only three high affinity nucleotide binding sites per hexamer are observed, the low affinity binding sites being nearly undetectable (Hingorani et al., 1996). The conversion of NTP to NDP and phosphate by helicase hydrolysis is necessary for helicase movement and DNA unwinding. Generally, Mg2+ is necessary for NTP hydrolysis. DNA or RNA greatly stimulates NTP hydrolysis from 10 to 100 fold. The Km for NTP ranges from a few micromolar to a few millimolar, and the stimulated kcat is determined to be as low as 0.6 s-1 and as high as 30 s-1 (Patel et al., 2000). To understand how the NTPase reaction at the catalytic sites of helicases is coupled to movement along and unwinding of dsDNA, it is import to obtain a complete description of the NTPase pathway and it is necessary to identify the steps in the NTPase reaction that lead to nucleic acid duplex binding, release, movement, and unwinding (Patel et al., 2000).